DNA antibody constructs for use against middle east respiratory syndrome coronavirus

ABSTRACT

Disclosed herein is a composition including a recombinant nucleic acid sequence that encodes an antibody to a Middle East Respiratory Syncytial Coronavirus (MERS-CoV) viral antigen. Also disclosed herein is a method of generating a synthetic antibody in a subject by administering the composition to the subject. The disclosure also provides a method of preventing and/or treating an MERS-CoV virus infection in a subject using said composition and method of generation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. national phase application filed under 35U.S.C. § 371 claiming priority to International Patent Application No.PCT/US18/53044, filed Sep. 27, 2018, which is entitled to priority under35 U.S.C. § 119(e) to U.S. Provisional Application No. 62/564,177, filedSep. 27, 2017, the contents of each of which are incorporated byreference herein in their entireties.

TECHNICAL FIELD

The present invention relates to a composition comprising a recombinantnucleic acid sequence for generating one or more synthetic antibodies,and functional fragments thereof, in vivo, and a method of preventingand/or treating Middle East Respiratory Syndrome coronavirus (MERS-CoV)infection in a subject by administering said composition.

BACKGROUND

Coronaviruses (CoV) are a family of viruses that are common worldwideand cause a range of illnesses in humans from the common cold to severeacute respiratory syndrome (SARS). Coronaviruses can also cause a numberof diseases in animals. Human coronaviruses 229E, OC43, NL63, and HKU1are endemic in the human population.

In 2012, a novel coronavirus (nCoV) emerged in Saudi Arabia and is nowknown as Middle East Respiratory Syndrome coronavirus (MERS-CoV).MERS-CoV can be classified as a beta coronavirus. Subsequent cases ofMERS-CoV infection have been reported elsewhere in the Middle East(e.g., Qatar and Jordan) and more recently in Europe. Infection withMERS-CoV presented as severe acute respiratory illness with symptoms offever, cough, and shortness of breath. About half of reported cases ofMERS-CoV infection have resulted in death and a majority of reportedcases have occurred in older to middle age men. Only a small number ofreported cases involved subjects with mild respiratory illness. Human tohuman transmission of MERS-CoV is possible, but very limited at thistime.

Thus, there is need in the art for improved therapeutics that preventand/or treat MERS-CoV infection. The current invention satisfies thisneed.

SUMMARY OF THE INVENTION

In one embodiment, the invention relates to a nucleic acid moleculeencoding one or more synthetic antibodies, wherein the nucleic acidmolecule comprises at least one of: a) a nucleotide sequence encoding ananti-Middle East Respiratory Syncytial Coronavirus (MERS-CoV) syntheticantibody; and b) a nucleotide sequence encoding a fragment of ananti-MERS-CoV synthetic antibody.

In one embodiment, the one or more synthetic antibodies binds to aMERS-CoV antigen.

In one embodiment, the antigen is MERS-CoV spike, MERS-CoV RNApolymerase, MERS-CoV nucleocapsid protein, MERS-CoV envelope protein,MERS-CoV matrix protein, any fragment thereof, or any combinationthereof.

In one embodiment, the nucleic acid molecule further comprises anucleotide sequence encoding a cleavage domain.

In one embodiment, the nucleic acid molecule comprises a nucleotidesequence encoding an anti-MERS-CoV antibody. In one embodiment, thenucleic acid molecule comprises a nucleotide sequence encoding an aminoacid sequence at least 90% homologous to SEQ ID NO:2. In one embodiment,the nucleic acid molecule comprises a nucleotide sequence at least 90%identical to SEQ ID NO:3.

In one embodiment, the nucleotide sequence encodes a leader sequence.

In one embodiment, the nucleic acid molecule is an expression vector.

In one embodiment, the invention relates to a composition comprising anucleotide sequence encoding an anti-MERS-CoV antibody. In oneembodiment, the composition further comprises a pharmaceuticallyacceptable excipient.

In one embodiment, the invention relates to a method of preventing ortreating a disease in a subject, the method comprising administering tothe subject a nucleic acid molecule comprising a nucleotide sequenceencoding an anti-MERS-CoV antibody or a composition comprising anucleotide sequence encoding an anti-MERS-CoV antibody.

In one embodiment, the disease is an MERS-CoV virus infection.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1, comprising FIG. 1A through FIG. 1D, depicts results from exampleexperiments demonstrating that delivery optimization enhances dMAb™expression in mice. (FIG. 1A) 25 μg pRFP was delivered into BALB/c mouseTA muscles with CELLECTRA®-3P and an enhanced formulation. The controlgroup received 25 μg pRFP without EP. TA muscles were harvested 72 hoursafter administration. The same delivery protocol described for (FIG. 1A)was employed for (FIG. 1B & FIG. 1C). (FIG. 1B) 100 μg dMAb-MERS or pVax(empty plasmid) were administered into BALB/c mouse TA muscles. IFimages of hIgG detected with anti-human IgG-FITC (green) are depictedwith DAPI stain (blue). (FIG. 1C) 6.25 to 100 μg of dMAb-MERS wasadministered into the TA muscle of Crl:Nu-Foxnlnu mice. Serum hIgGlevels were quantified by ELISA. (FIG. 1D) 100 μg dMAb-MERS wasdelivered to BALB/c mouse TA muscle using delivery protocol (FIG. 1A)—EPand enhanced formulation (blue line) or enhanced formulation without EP(black line). Binding of serum hIgG to MERS CoV antigen was measured byELISA 6 days after dMAb-MERS delivery.

FIG. 2, comprising FIG. 2A and FIG. 2B, depicts results from exampleexperiments demonstrating in vivo dMAb™ expression in rhesus macaques.13.5 mg of dMAb-pMERS was administered with EP into the quad muscles ofrhesus macaques employing the CELLECTRA®-5P delivery system and anenhanced formulation. Serum hIgG was quantified (FIG. 2A) and MERS CoVantigen binding measured by ELISA at intervals between days 0 to 35(FIG. 2B). Individual values of 6 rhesus macaques.

FIG. 3 depicts a plasmid map of an expression plasmid encoding dMAb-MERS(pGX9207; SEQ ID NO:1).

DETAILED DESCRIPTION

The present invention relates to compositions comprising a recombinantnucleic acid sequence encoding an antibody, a fragment thereof, avariant thereof, or a combination thereof. The composition can beadministered to a subject in need thereof to facilitate in vivoexpression and formation of a synthetic antibody.

In one embodiment, the heavy chain and light chain polypeptidesexpressed from the recombinant nucleic acid sequences can assemble intothe synthetic antibody. The heavy chain polypeptide and the light chainpolypeptide can interact with one another such that assembly results inthe synthetic antibody being capable of binding the antigen, being moreimmunogenic as compared to an antibody not assembled as describedherein, and being capable of eliciting or inducing an immune responseagainst the antigen.

Additionally, these synthetic antibodies are generated more rapidly inthe subject than antibodies that are produced in response to antigeninduced immune response. The synthetic antibodies are able toeffectively bind and neutralize a range of antigens. The syntheticantibodies are also able to effectively protect against and/or promotesurvival of disease.

1. Definitions

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. In case of conflict, the present document, includingdefinitions, will control. Preferred methods and materials are describedbelow, although methods and materials similar or equivalent to thosedescribed herein can be used in practice or testing of the presentinvention. All publications, patent applications, patents and otherreferences mentioned herein are incorporated by reference in theirentirety. The materials, methods, and examples disclosed herein areillustrative only and not intended to be limiting.

The terms “comprise(s),” “include(s),” “having,” “has,” “can,”“contain(s),” and variants thereof, as used herein, are intended to beopen-ended transitional phrases, terms, or words that do not precludethe possibility of additional acts or structures. The singular forms“a,” “and” and “the” include plural references unless the contextclearly dictates otherwise. The present disclosure also contemplatesother embodiments, “comprising,” “consisting of” and “consistingessentially of,” the embodiments, or elements presented herein, whetherexplicitly set forth or not.

“Antibody” may mean an antibody of classes IgG, IgM, IgA, IgD or IgE, orfragments, fragments or derivatives thereof, including Fab, F(ab′)2, Fd,and single chain antibodies, and derivatives thereof. The antibody maybe an antibody isolated from the serum sample of mammal, a polyclonalantibody, affinity purified antibody, or mixtures thereof which exhibitssufficient binding specificity to a desired epitope or a sequencederived therefrom.

“Antibody fragment” or “fragment of an antibody” as used interchangeablyherein refers to a portion of an intact antibody comprising theantigen-binding site or variable region. The portion does not includethe constant heavy chain domains (i.e. CH2, CH3, or CH4, depending onthe antibody isotype) of the Fc region of the intact antibody. Examplesof antibody fragments include, but are not limited to, Fab fragments,Fab′ fragments, Fab′-SH fragments, F(ab′)2 fragments, Fd fragments, Fvfragments, diabodies, single-chain Fv (scFv) molecules, single-chainpolypeptides containing only one light chain variable domain,single-chain polypeptides containing the three CDRs of the light-chainvariable domain, single-chain polypeptides containing only one heavychain variable region, and single-chain polypeptides containing thethree CDRs of the heavy chain variable region.

“Antigen” refers to proteins that have the ability to generate an immuneresponse in a host. An antigen may be recognized and bound by anantibody. An antigen may originate from within the body or from theexternal environment.

“CDRs” are defined as the complementarity determining region amino acidsequences of an antibody which are the hypervariable regions ofimmunoglobulin heavy and light chains. See, e.g., Kabat et al.,Sequences of Proteins of Immunological Interest, 4th Ed., U.S.Department of Health and Human Services, National Institutes of Health(1987). There are three heavy chain and three light chain CDRs (or CDRregions) in the variable portion of an immunoglobulin. Thus, “CDRs” asused herein refers to all three heavy chain CDRs, or all three lightchain CDRs (or both all heavy and all light chain CDRs, if appropriate).The structure and protein folding of the antibody may mean that otherresidues are considered part of the antigen binding region and would beunderstood to be so by a skilled person. See for example Chothia et al.,(1989) Conformations of immunoglobulin hypervariable regions; Nature342, p 877-883.

“Coding sequence” or “encoding nucleic acid” as used herein may meanrefers to the nucleic acid (RNA or DNA molecule) that comprise anucleotide sequence which encodes an antibody as set forth herein. Thecoding sequence may also comprise a DNA sequence which encodes an RNAsequence. The coding sequence may further include initiation andtermination signals operably linked to regulatory elements including apromoter and polyadenylation signal capable of directing expression inthe cells of an individual or mammal to whom the nucleic acid isadministered. The coding sequence may further include sequences thatencode signal peptides.

“Complement” or “complementary” as used herein may mean a nucleic acidmay mean Watson-Crick (e.g., A-T/U and C-G) or Hoogsteen base pairingbetween nucleotides or nucleotide analogs of nucleic acid molecules.

“Constant current” as used herein to define a current that is receivedor experienced by a tissue, or cells defining said tissue, over theduration of an electrical pulse delivered to same tissue. The electricalpulse is delivered from the electroporation devices described herein.This current remains at a constant amperage in said tissue over the lifeof an electrical pulse because the electroporation device providedherein has a feedback element, preferably having instantaneous feedback.The feedback element can measure the resistance of the tissue (or cells)throughout the duration of the pulse and cause the electroporationdevice to alter its electrical energy output (e.g., increase voltage) socurrent in same tissue remains constant throughout the electrical pulse(on the order of microseconds), and from pulse to pulse. In someembodiments, the feedback element comprises a controller.

“Current feedback” or “feedback” as used herein may be usedinterchangeably and may mean the active response of the providedelectroporation devices, which comprises measuring the current in tissuebetween electrodes and altering the energy output delivered by the EPdevice accordingly in order to maintain the current at a constant level.This constant level is preset by a user prior to initiation of a pulsesequence or electrical treatment. The feedback may be accomplished bythe electroporation component, e.g., controller, of the electroporationdevice, as the electrical circuit therein is able to continuouslymonitor the current in tissue between electrodes and compare thatmonitored current (or current within tissue) to a preset current andcontinuously make energy-output adjustments to maintain the monitoredcurrent at preset levels. The feedback loop may be instantaneous as itis an analog closed-loop feedback.

“Decentralized current” as used herein may mean the pattern ofelectrical currents delivered from the various needle electrode arraysof the electroporation devices described herein, wherein the patternsminimize, or preferably eliminate, the occurrence of electroporationrelated heat stress on any area of tissue being electroporated.

“Electroporation,” “electro-permeabilization,” or “electro-kineticenhancement” (“EP”) as used interchangeably herein may refer to the useof a transmembrane electric field pulse to induce microscopic pathways(pores) in a bio-membrane; their presence allows biomolecules such asplasmids, oligonucleotides, siRNA, drugs, ions, and water to pass fromone side of the cellular membrane to the other.

“Endogenous antibody” as used herein may refer to an antibody that isgenerated in a subject that is administered an effective dose of anantigen for induction of a humoral immune response.

“Feedback mechanism” as used herein may refer to a process performed byeither software or hardware (or firmware), which process receives andcompares the impedance of the desired tissue (before, during, and/orafter the delivery of pulse of energy) with a present value, preferablycurrent, and adjusts the pulse of energy delivered to achieve the presetvalue. A feedback mechanism may be performed by an analog closed loopcircuit.

“Fragment” may mean a polypeptide fragment of an antibody that isfunction, i.e., can bind to desired target and have the same intendedeffect as a full length antibody. A fragment of an antibody may be 100%identical to the full length except missing at least one amino acid fromthe N and/or C terminal, in each case with or without signal peptidesand/or a methionine at position 1. Fragments may comprise 20% or more,25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% ormore, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more,80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% ormore, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more,99% or more percent of the length of the particular full lengthantibody, excluding any heterologous signal peptide added. The fragmentmay comprise a fragment of a polypeptide that is 95% or more, 96% ormore, 97% or more, 98% or more or 99% or more identical to the antibodyand additionally comprise an N terminal methionine or heterologoussignal peptide which is not included when calculating percent identity.Fragments may further comprise an N terminal methionine and/or a signalpeptide such as an immunoglobulin signal peptide, for example an IgE orIgG signal peptide. The N terminal methionine and/or signal peptide maybe linked to a fragment of an antibody.

A fragment of a nucleic acid sequence that encodes an antibody may be100% identical to the full length except missing at least one nucleotidefrom the 5′ and/or 3′ end, in each case with or without sequencesencoding signal peptides and/or a methionine at position 1. Fragmentsmay comprise 20% or more, 25% or more, 30% or more, 35% or more, 40% ormore, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more,70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% ormore, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more,97% or more, 98% or more, 99% or more percent of the length of theparticular full length coding sequence, excluding any heterologoussignal peptide added. The fragment may comprise a fragment that encode apolypeptide that is 95% or more, 96% or more, 97% or more, 98% or moreor 99% or more identical to the antibody and additionally optionallycomprise sequence encoding an N terminal methionine or heterologoussignal peptide which is not included when calculating percent identity.Fragments may further comprise coding sequences for an N terminalmethionine and/or a signal peptide such as an immunoglobulin signalpeptide, for example an IgE or IgG signal peptide. The coding sequenceencoding the N terminal methionine and/or signal peptide may be linkedto a fragment of coding sequence.

In one embodiment, the invention relates to nucleic acid moleculesencoding a fragment of an anti-MERS-CoV antibody. Fragments of anantibody include, but are not limited to, fragments comprising a singlechain (e.g., a heavy chain or a light chain) of the antibody. In oneembodiment, the fragment of an antibody may comprise a heavy chainfragment of an anti-MERS-CoV antibody. In one embodiment, a heavy chainfragment of an anti-MERS-CoV antibody comprises an amino acid sequenceas set forth in SEQ ID NO:5. Therefore, in one embodiment, thenucleotide sequence encoding a fragment of an anti-MERS-CoV antibodycomprises one or more codon optimized nucleic acid sequences encoding anamino acid sequence at least 90% homologous to the amino acid sequenceof SEQ ID NO:5, or a fragment of an amino acid sequence at least 90%homologous to the amino acid sequence of SEQ ID NO:5. In one embodiment,the nucleotide sequence encoding an anti-MERS-CoV antibody comprises oneor more codon optimized nucleic acid sequences encoding the amino acidsequence of SEQ ID NO:5 or a fragment of the amino acid sequence of SEQID NO:5.

In one embodiment, the nucleotide sequence encoding a fragment of ananti-MERS-CoV antibody comprises one or more RNA sequences transcribedfrom one or more DNA sequences encoding a fragment of an amino acidsequence at least 90% homologous to SEQ ID NO:5 or a fragment of anamino acid sequence at least 90% homologous to SEQ ID NO:5.

In one embodiment, the nucleotide sequence encoding a fragment of ananti-MERS-CoV antibody comprises one or more codon optimized nucleicacid sequences at least 90% homologous to SEQ ID NO:4 or a fragment of anucleic acid sequence at least 90% homologous to SEQ ID NO:4. In oneembodiment, the nucleotide sequence encoding an anti-MERS-CoV antibodycomprises one or more codon optimized nucleic acid sequences as setforth in SEQ ID NO:4 or a fragment of a nucleic acid sequence as setforth in SEQ ID NO:4.

In one embodiment, the nucleotide sequence encoding a fragment of ananti-MERS-CoV antibody comprises one or more RNA sequences transcribedfrom one or more DNA sequences at least 90% homologous to SEQ ID NO:4 ora fragment of a DNA sequence at least 90% homologous to SEQ ID NO:4.

In one embodiment, the fragment of an antibody may comprise a lightchain fragment of an anti-MERS-CoV antibody. In one embodiment, a lightchain fragment of an anti-MERS-CoV antibody comprises an amino acidsequence as set forth in SEQ ID NO:7. Therefore, in one embodiment, thenucleotide sequence encoding a fragment of an anti-MERS-CoV antibodycomprises one or more codon optimized nucleic acid sequences encoding anamino acid sequence at least 90% homologous to the amino acid sequenceof SEQ ID NO:7, or a fragment of an amino acid sequence at least 90%homologous to the amino acid sequence of SEQ ID NO:7. In one embodiment,the nucleotide sequence encoding an anti-MERS-CoV antibody comprises oneor more codon optimized nucleic acid sequences encoding the amino acidsequence of SEQ ID NO:7 or a fragment of the amino acid sequence of SEQID NO:7.

In one embodiment, the nucleotide sequence encoding a fragment of ananti-MERS-CoV antibody comprises one or more RNA sequences transcribedfrom one or more DNA sequences encoding a fragment of an amino acidsequence at least 90% homologous to SEQ ID NO:7 or a fragment of anamino acid sequence at least 90% homologous to SEQ ID NO:7.

In one embodiment, the nucleotide sequence encoding a fragment of ananti-MERS-CoV antibody comprises one or more codon optimized nucleicacid sequences at least 90% homologous to SEQ ID NO:6 or a fragment of anucleic acid sequence at least 90% homologous to SEQ ID NO:6. In oneembodiment, the nucleotide sequence encoding an anti-MERS-CoV antibodycomprises one or more codon optimized nucleic acid sequences as setforth in SEQ ID NO:6 or a fragment of a nucleic acid sequence as setforth in SEQ ID NO:6.

In one embodiment, the nucleotide sequence encoding a fragment of ananti-MERS-CoV antibody comprises one or more RNA sequences transcribedfrom one or more DNA sequences at least 90% homologous to SEQ ID NO:6 ora fragment of a DNA sequence at least 90% homologous to SEQ ID NO:6.

“Genetic construct” as used herein refers to the DNA or RNA moleculesthat comprise a nucleotide sequence which encodes a protein, such as anantibody. The genetic construct may also refer to a DNA molecule whichtranscribes an RNA. The coding sequence includes initiation andtermination signals operably linked to regulatory elements including apromoter and polyadenylation signal capable of directing expression inthe cells of the individual to whom the nucleic acid molecule isadministered. As used herein, the term “expressible form” refers to geneconstructs that contain the necessary regulatory elements operablelinked to a coding sequence that encodes a protein such that whenpresent in the cell of the individual, the coding sequence will beexpressed.

“Identical” or “identity” as used herein in the context of two or morenucleic acids or polypeptide sequences, may mean that the sequences havea specified percentage of residues that are the same over a specifiedregion. The percentage may be calculated by optimally aligning the twosequences, comparing the two sequences over the specified region,determining the number of positions at which the identical residueoccurs in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the specified region, and multiplying the result by 100 toyield the percentage of sequence identity. In cases where the twosequences are of different lengths or the alignment produces one or morestaggered ends and the specified region of comparison includes only asingle sequence, the residues of single sequence are included in thedenominator but not the numerator of the calculation. When comparing DNAand RNA, thymine (T) and uracil (U) may be considered equivalent.Identity may be performed manually or by using a computer sequencealgorithm such as BLAST or BLAST 2.0.

“Impedance” as used herein may be used when discussing the feedbackmechanism and can be converted to a current value according to Ohm'slaw, thus enabling comparisons with the preset current.

“Immune response” as used herein may mean the activation of a host'simmune system, e.g., that of a mammal, in response to the introductionof one or more nucleic acids and/or peptides. The immune response can bein the form of a cellular or humoral response, or both.

“Nucleic acid” or “oligonucleotide” or “polynucleotide” as used hereinmay mean at least two nucleotides covalently linked together. Thedepiction of a single strand also defines the sequence of thecomplementary strand. Thus, a nucleic acid also encompasses thecomplementary strand of a depicted single strand. Many variants of anucleic acid may be used for the same purpose as a given nucleic acid.Thus, a nucleic acid also encompasses substantially identical nucleicacids and complements thereof. A single strand provides a probe that mayhybridize to a target sequence under stringent hybridization conditions.Thus, a nucleic acid also encompasses a probe that hybridizes understringent hybridization conditions.

Nucleic acids may be single stranded or double stranded, or may containportions of both double stranded and single stranded sequence. Thenucleic acid may be DNA, both genomic and cDNA, RNA, or a hybrid, wherethe nucleic acid may contain combinations of deoxyribo- andribo-nucleotides, and combinations of bases including uracil, adenine,thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosineand isoguanine. Nucleic acids may be obtained by chemical synthesismethods or by recombinant methods.

“Operably linked” as used herein may mean that expression of a gene isunder the control of a promoter with which it is spatially connected. Apromoter may be positioned 5′ (upstream) or 3′ (downstream) of a geneunder its control. The distance between the promoter and a gene may beapproximately the same as the distance between that promoter and thegene it controls in the gene from which the promoter is derived. As isknown in the art, variation in this distance may be accommodated withoutloss of promoter function.

A “peptide,” “protein,” or “polypeptide” as used herein can mean alinked sequence of amino acids and can be natural, synthetic, or amodification or combination of natural and synthetic.

“Promoter” as used herein may mean a synthetic or naturally-derivedmolecule which is capable of conferring, activating or enhancingexpression of a nucleic acid in a cell. A promoter may comprise one ormore specific transcriptional regulatory sequences to further enhanceexpression and/or to alter the spatial expression and/or temporalexpression of same. A promoter may also comprise distal enhancer orrepressor elements, which can be located as much as several thousandbase pairs from the start site of transcription. A promoter may bederived from sources including viral, bacterial, fungal, plants,insects, and animals. A promoter may regulate the expression of a genecomponent constitutively, or differentially with respect to cell, thetissue or organ in which expression occurs or, with respect to thedevelopmental stage at which expression occurs, or in response toexternal stimuli such as physiological stresses, pathogens, metal ions,or inducing agents. Representative examples of promoters include thebacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lacoperator-promoter, tac promoter, SV40 late promoter, SV40 earlypromoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter or SV40 late promoter and the CMV IE promoter.

“Signal peptide” and “leader sequence” are used interchangeably hereinand refer to an amino acid sequence that can be linked at the aminoterminus of a protein set forth herein. Signal peptides/leader sequencestypically direct localization of a protein. Signal peptides/leadersequences used herein preferably facilitate secretion of the proteinfrom the cell in which it is produced. Signal peptides/leader sequencesare often cleaved from the remainder of the protein, often referred toas the mature protein, upon secretion from the cell. Signalpeptides/leader sequences are linked at the N terminus of the protein.

“Stringent hybridization conditions” as used herein may mean conditionsunder which a first nucleic acid sequence (e.g., probe) will hybridizeto a second nucleic acid sequence (e.g., target), such as in a complexmixture of nucleic acids. Stringent conditions are sequence dependentand will be different in different circumstances. Stringent conditionsmay be selected to be about 5-10° C. lower than the thermal meltingpoint (T_(m)) for the specific sequence at a defined ionic strength pH.The T_(m) may be the temperature (under defined ionic strength, pH, andnucleic concentration) at which 50% of the probes complementary to thetarget hybridize to the target sequence at equilibrium (as the targetsequences are present in excess, at T_(m), 50% of the probes areoccupied at equilibrium). Stringent conditions may be those in which thesalt concentration is less than about 1.0 M sodium ion, such as about0.01-1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3and the temperature is at least about 30° C. for short probes (e.g.,about 10-50 nucleotides) and at least about 60° C. for long probes(e.g., greater than about 50 nucleotides). Stringent conditions may alsobe achieved with the addition of destabilizing agents such as formamide.For selective or specific hybridization, a positive signal may be atleast 2 to 10 times background hybridization. Exemplary stringenthybridization conditions include the following: 50% formamide, 5×SSC,and 1% SDS, incubating at 42° C., or, 5×SSC, 1% SDS, incubating at 65°C., with wash in 0.2×SSC, and 0.1% SDS at 65° C.

“Subject” and “patient” as used herein interchangeably refers to anyvertebrate, including, but not limited to, a mammal (e.g., cow, pig,camel, llama, horse, goat, rabbit, sheep, hamsters, guinea pig, cat,dog, rat, and mouse, a non-human primate (for example, a monkey, such asa cynomolgous or rhesus monkey, chimpanzee, etc) and a human). In someembodiments, the subject may be a human or a non-human. The subject orpatient may be undergoing other forms of treatment.

“Substantially complementary” as used herein may mean that a firstsequence is at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to the complement of a second sequence over a region of 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more nucleotidesor amino acids, or that the two sequences hybridize under stringenthybridization conditions.

“Substantially identical” as used herein may mean that a first andsecond sequence are at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% over a region of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800,900, 1000, 1100 or more nucleotides or amino acids, or with respect tonucleic acids, if the first sequence is substantially complementary tothe complement of the second sequence.

“Synthetic antibody” as used herein refers to an antibody that isencoded by the recombinant nucleic acid sequence described herein and isgenerated in a subject.

“Treatment” or “treating,” as used herein can mean protecting of asubject from a disease through means of preventing, suppressing,repressing, or completely eliminating the disease. Preventing thedisease involves administering a vaccine of the present invention to asubject prior to onset of the disease. Suppressing the disease involvesadministering a vaccine of the present invention to a subject afterinduction of the disease but before its clinical appearance. Repressingthe disease involves administering a vaccine of the present invention toa subject after clinical appearance of the disease.

“Variant” used herein with respect to a nucleic acid may mean (i) aportion or fragment of a referenced nucleotide sequence; (ii) thecomplement of a referenced nucleotide sequence or portion thereof; (iii)a nucleic acid that is substantially identical to a referenced nucleicacid or the complement thereof; or (iv) a nucleic acid that hybridizesunder stringent conditions to the referenced nucleic acid, complementthereof, or a sequences substantially identical thereto.

“Variant” with respect to a peptide or polypeptide that differs in aminoacid sequence by the insertion, deletion, or conservative substitutionof amino acids, but retain at least one biological activity. Variant mayalso mean a protein with an amino acid sequence that is substantiallyidentical to a referenced protein with an amino acid sequence thatretains at least one biological activity. A conservative substitution ofan amino acid, i.e., replacing an amino acid with a different amino acidof similar properties (e.g., hydrophilicity, degree and distribution ofcharged regions) is recognized in the art as typically involving a minorchange. These minor changes can be identified, in part, by consideringthe hydropathic index of amino acids, as understood in the art. Kyte etal., J. Mol. Biol. 157:105-132 (1982). The hydropathic index of an aminoacid is based on a consideration of its hydrophobicity and charge. It isknown in the art that amino acids of similar hydropathic indexes can besubstituted and still retain protein function. In one aspect, aminoacids having hydropathic indexes of ±2 are substituted. Thehydrophilicity of amino acids can also be used to reveal substitutionsthat would result in proteins retaining biological function. Aconsideration of the hydrophilicity of amino acids in the context of apeptide permits calculation of the greatest local average hydrophilicityof that peptide, a useful measure that has been reported to correlatewell with antigenicity and immunogenicity. U.S. Pat. No. 4,554,101,incorporated fully herein by reference. Substitution of amino acidshaving similar hydrophilicity values can result in peptides retainingbiological activity, for example immunogenicity, as is understood in theart. Substitutions may be performed with amino acids havinghydrophilicity values within ±2 of each other. Both the hyrophobicityindex and the hydrophilicity value of amino acids are influenced by theparticular side chain of that amino acid. Consistent with thatobservation, amino acid substitutions that are compatible withbiological function are understood to depend on the relative similarityof the amino acids, and particularly the side chains of those aminoacids, as revealed by the hydrophobicity, hydrophilicity, charge, size,and other properties.

A variant may be a nucleic acid sequence that is substantially identicalover the full length of the full gene sequence or a fragment thereof.The nucleic acid sequence may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical over the full length of the gene sequence or a fragmentthereof. A variant may be an amino acid sequence that is substantiallyidentical over the full length of the amino acid sequence or fragmentthereof. The amino acid sequence may be 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% identical over the full length of the amino acid sequence or afragment thereof.

“Vector” as used herein may mean a nucleic acid sequence containing anorigin of replication. A vector may be a plasmid, bacteriophage,bacterial artificial chromosome or yeast artificial chromosome. A vectormay be a DNA or RNA vector. A vector may be either a self-replicatingextrachromosomal vector or a vector which integrates into a host genome.

For the recitation of numeric ranges herein, each intervening numberthere between with the same degree of precision is explicitlycontemplated. For example, for the range of 6-9, the numbers 7 and 8 arecontemplated in addition to 6 and 9, and for the range 6.0-7.0, thenumber 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 areexplicitly contemplated.

2. Composition

The present invention relates to a composition comprising a recombinantnucleic acid sequence encoding an antibody, a fragment thereof, avariant thereof, or a combination thereof. The composition, whenadministered to a subject in need thereof, can result in the generationof a synthetic antibody in the subject. The synthetic antibody can binda target molecule (i.e., an antigen) present in the subject. Suchbinding can neutralize the antigen, block recognition of the antigen byanother molecule, for example, a protein or nucleic acid, and elicit orinduce an immune response to the antigen.

In one embodiment, the composition comprises a nucleotide sequenceencoding a synthetic antibody. In one embodiment, the compositioncomprises a nucleic acid molecule comprising a first nucleotide sequenceencoding a first synthetic antibody and a second nucleotide sequenceencoding a second synthetic antibody. In one embodiment, the nucleicacid molecule comprises a nucleotide sequence encoding a cleavagedomain.

In one embodiment, the nucleic acid molecule comprises a nucleotidesequence encoding an anti-Middle East Respiratory Syndrome coronavirus(MERS-CoV) (anti-MERS-CoV) antibody.

In one embodiment, the nucleotide sequence encoding an anti-MERS-CoVantibody comprises one or more codon optimized nucleic acid sequencesencoding an amino acid sequence at least 90% homologous to the aminoacid sequence of SEQ ID NO:2, or a fragment of an amino acid sequence atleast 90% homologous to the amino acid sequence of SEQ ID NO:2. In oneembodiment, the nucleotide sequence encoding an anti-MERS-CoV antibodycomprises one or more codon optimized nucleic acid sequences encodingthe amino acid sequence of SEQ ID NO:2 or a fragment of the amino acidsequence of SEQ ID NO:2.

In one embodiment, the nucleotide sequence encoding an anti-MERS-CoVantibody comprises one or more RNA sequences transcribed from one ormore DNA sequences encoding an amino acid sequence at least 90%homologous to SEQ ID NO:2 or a fragment of an amino acid sequence atleast 90% homologous to SEQ ID NO:2. In one embodiment, the nucleotidesequence encoding an anti-MERS-CoV antibody comprises one or more RNAsequences transcribed from one or more DNA sequences encoding an aminoacid sequence as set forth in SEQ ID NO:2 or a fragment of an amino acidsequence as set forth in SEQ ID NO:2.

In one embodiment, the nucleotide sequence encoding an anti-MERS-CoVantibody comprises one or more codon optimized nucleic acid sequences atleast 90% homologous to SEQ ID NO:3 or a fragment of a nucleic acidsequence at least 90% homologous to SEQ ID NO:3. In one embodiment, thenucleotide sequence encoding an anti-MERS-CoV antibody comprises one ormore codon optimized nucleic acid sequences as set forth in SEQ ID NO:3or a fragment of a nucleic acid sequence as set forth in SEQ ID NO:3.

In one embodiment, the nucleotide sequence encoding an anti-MERS-CoVantibody comprises one or more RNA sequences transcribed from one ormore DNA sequences at least 90% homologous to SEQ ID NO:3 or a fragmentof a DNA sequence at least 90% homologous to SEQ ID NO:3. In oneembodiment, the nucleotide sequence encoding an anti-MERS-CoV antibodycomprises one or more RNA sequence transcribed from one or more DNAsequences as set forth in SEQ ID NO:3 or a fragment of a DNA sequence asset forth in SEQ ID NO:3.

In one embodiment, the nucleic acid molecule comprising a nucleotidesequence encoding an anti-Middle East Respiratory Syndrome coronaviruscomprises the nucleotide sequence as set forth in SEQ ID NO:1,comprising a nucleotide sequence encoding an anti-MERS-CoV antibody inan expression plasmid for expression in humans.

The composition of the invention can treat, prevent and/or protectagainst any disease, disorder, or condition associated with MERS-CoVvirus infection. In certain embodiments, the composition can treat,prevent, and or/protect against viral infection. In certain embodiments,the composition can treat, prevent, and or/protect against a conditionassociated with MERS-CoV virus infection.

3. Recombinant Nucleic Acid Sequence

As described above, the composition can comprise a recombinant nucleicacid sequence. The recombinant nucleic acid sequence can encode theantibody, a fragment thereof, a variant thereof, or a combinationthereof. The antibody is described in more detail below.

The recombinant nucleic acid sequence can be a heterologous nucleic acidsequence. The recombinant nucleic acid sequence can include one or moreheterologous nucleic acid sequences.

The recombinant nucleic acid sequence can be an optimized nucleic acidsequence. Such optimization can increase or alter the immunogenicity ofthe antibody. Optimization can also improve transcription and/ortranslation. Optimization can include one or more of the following: lowGC content leader sequence to increase transcription; mRNA stability andcodon optimization; addition of a kozak sequence (e.g., GCC ACC) forincreased translation; addition of an immunoglobulin (Ig) leadersequence encoding a signal peptide; addition of an internal IRESsequence and eliminating to the extent possible cis-acting sequencemotifs (i.e., internal TATA boxes).

Recombinant Nucleic Acid Sequence Construct

The recombinant nucleic acid sequence can include one or morerecombinant nucleic acid sequence constructs. The recombinant nucleicacid sequence construct can include one or more components, which aredescribed in more detail below.

The recombinant nucleic acid sequence construct can include aheterologous nucleic acid sequence that encodes a heavy chainpolypeptide, a fragment thereof, a variant thereof, or a combinationthereof. The recombinant nucleic acid sequence construct can include aheterologous nucleic acid sequence that encodes a light chainpolypeptide, a fragment thereof, a variant thereof, or a combinationthereof. The recombinant nucleic acid sequence construct can alsoinclude a heterologous nucleic acid sequence that encodes a protease orpeptidase cleavage site. The recombinant nucleic acid sequence constructcan also include a heterologous nucleic acid sequence that encodes aninternal ribosome entry site (IRES). An IRES may be either a viral IRESor a eukaryotic IRES. The recombinant nucleic acid sequence constructcan include one or more leader sequences, in which each leader sequenceencodes a signal peptide. The recombinant nucleic acid sequenceconstruct can include one or more promoters, one or more introns, one ormore transcription termination regions, one or more initiation codons,one or more termination or stop codons, and/or one or morepolyadenylation signals. The recombinant nucleic acid sequence constructcan also include one or more linker or tag sequences. The tag sequencecan encode a hemagglutinin (HA) tag.

(1) Heavy Chain Polypeptide

The recombinant nucleic acid sequence construct can include theheterologous nucleic acid encoding the heavy chain polypeptide, afragment thereof, a variant thereof, or a combination thereof. The heavychain polypeptide can include a variable heavy chain (VH) region and/orat least one constant heavy chain (CH) region. The at least one constantheavy chain region can include a constant heavy chain region 1 (CH1), aconstant heavy chain region 2 (CH2), and a constant heavy chain region 3(CH3), and/or a hinge region.

In some embodiments, the heavy chain polypeptide can include a VH regionand a CH1 region. In other embodiments, the heavy chain polypeptide caninclude a VH region, a CH1 region, a hinge region, a CH2 region, and aCH3 region.

The heavy chain polypeptide can include a complementarity determiningregion (“CDR”) set. The CDR set can contain three hypervariable regionsof the VH region. Proceeding from N-terminus of the heavy chainpolypeptide, these CDRs are denoted “CDR1,” “CDR2,” and “CDR3,”respectively. CDR1, CDR2, and CDR3 of the heavy chain polypeptide cancontribute to binding or recognition of the antigen.

(2) Light Chain Polypeptide

The recombinant nucleic acid sequence construct can include theheterologous nucleic acid sequence encoding the light chain polypeptide,a fragment thereof, a variant thereof, or a combination thereof. Thelight chain polypeptide can include a variable light chain (VL) regionand/or a constant light chain (CL) region.

The light chain polypeptide can include a complementarity determiningregion (“CDR”) set. The CDR set can contain three hypervariable regionsof the VL region. Proceeding from N-terminus of the light chainpolypeptide, these CDRs are denoted “CDR1,” “CDR2,” and “CDR3,”respectively. CDR1, CDR2, and CDR3 of the light chain polypeptide cancontribute to binding or recognition of the antigen.

(3) Protease Cleavage Site

The recombinant nucleic acid sequence construct can include heterologousnucleic acid sequence encoding a protease cleavage site. The proteasecleavage site can be recognized by a protease or peptidase. The proteasecan be an endopeptidase or endoprotease, for example, but not limitedto, furin, elastase, HtrA, calpain, trypsin, chymotrypsin, trypsin, andpepsin. The protease can be furin. In other embodiments, the proteasecan be a serine protease, a threonine protease, cysteine protease,aspartate protease, metalloprotease, glutamic acid protease, or anyprotease that cleaves an internal peptide bond (i.e., does not cleavethe N-terminal or C-terminal peptide bond).

The protease cleavage site can include one or more amino acid sequencesthat promote or increase the efficiency of cleavage. The one or moreamino acid sequences can promote or increase the efficiency of formingor generating discrete polypeptides. The one or more amino acidssequences can include a 2A peptide sequence.

(4) Linker Sequence

The recombinant nucleic acid sequence construct can include one or morelinker sequences. The linker sequence can spatially separate or link theone or more components described herein. In other embodiments, thelinker sequence can encode an amino acid sequence that spatiallyseparates or links two or more polypeptides.

(5) Promoter

The recombinant nucleic acid sequence construct can include one or morepromoters. The one or more promoters may be any promoter that is capableof driving gene expression and regulating gene expression. Such apromoter is a cis-acting sequence element required for transcription viaa DNA dependent RNA polymerase. Selection of the promoter used to directgene expression depends on the particular application. The promoter maybe positioned about the same distance from the transcription start inthe recombinant nucleic acid sequence construct as it is from thetranscription start site in its natural setting. However, variation inthis distance may be accommodated without loss of promoter function.

The promoter may be operably linked to the heterologous nucleic acidsequence encoding the heavy chain polypeptide and/or light chainpolypeptide. The promoter may be a promoter shown effective forexpression in eukaryotic cells. The promoter operably linked to thecoding sequence may be a CMV promoter, a promoter from simian virus 40(SV40), such as SV40 early promoter and SV40 later promoter, a mousemammary tumor virus (MMTV) promoter, a human immunodeficiency virus(HIV) promoter such as the bovine immunodeficiency virus (BIV) longterminal repeat (LTR) promoter, a Moloney virus promoter, an avianleukosis virus (ALV) promoter, a cytomegalovirus (CMV) promoter such asthe CMV immediate early promoter, Epstein Barr virus (EBV) promoter, ora Rous sarcoma virus (RSV) promoter. The promoter may also be a promoterfrom a human gene such as human actin, human myosin, human hemoglobin,human muscle creatine, human polyhedrin, or human metalothionein.

The promoter can be a constitutive promoter or an inducible promoter,which initiates transcription only when the host cell is exposed to someparticular external stimulus. In the case of a multicellular organism,the promoter can also be specific to a particular tissue or organ orstage of development. The promoter may also be a tissue specificpromoter, such as a muscle or skin specific promoter, natural orsynthetic. Examples of such promoters are described in US patentapplication publication no. US20040175727, the contents of which areincorporated herein in its entirety.

The promoter can be associated with an enhancer. The enhancer can belocated upstream of the coding sequence. The enhancer may be humanactin, human myosin, human hemoglobin, human muscle creatine or a viralenhancer such as one from CMV, FMDV, RSV or EBV. Polynucleotide functionenhances are described in U.S. Pat. Nos. 5,593,972, 5,962,428, andWO94/016737, the contents of each are fully incorporated by reference.

(6) Intron

The recombinant nucleic acid sequence construct can include one or moreintrons. Each intron can include functional splice donor and acceptorsites. The intron can include an enhancer of splicing. The intron caninclude one or more signals required for efficient splicing.

(7) Transcription Termination Region

The recombinant nucleic acid sequence construct can include one or moretranscription termination regions. The transcription termination regioncan be downstream of the coding sequence to provide for efficienttermination. The transcription termination region can be obtained fromthe same gene as the promoter described above or can be obtained fromone or more different genes.

(8) Initiation Codon

The recombinant nucleic acid sequence construct can include one or moreinitiation codons. The initiation codon can be located upstream of thecoding sequence. The initiation codon can be in frame with the codingsequence. The initiation codon can be associated with one or moresignals required for efficient translation initiation, for example, butnot limited to, a ribosome binding site.

(9) Termination Codon

The recombinant nucleic acid sequence construct can include one or moretermination or stop codons. The termination codon can be downstream ofthe coding sequence. The termination codon can be in frame with thecoding sequence. The termination codon can be associated with one ormore signals required for efficient translation termination.

(10) Polyadenylation Signal

The recombinant nucleic acid sequence construct can include one or morepolyadenylation signals. The polyadenylation signal can include one ormore signals required for efficient polyadenylation of the transcript.The polyadenylation signal can be positioned downstream of the codingsequence. The polyadenylation signal may be a SV40 polyadenylationsignal, LTR polyadenylation signal, bovine growth hormone (bGH)polyadenylation signal, human growth hormone (hGH) polyadenylationsignal, or human β-globin polyadenylation signal. The SV40polyadenylation signal may be a polyadenylation signal from a pCEP4plasmid (Invitrogen, San Diego, Calif.).

(11) Leader Sequence

The recombinant nucleic acid sequence construct can include one or moreleader sequences. The leader sequence can encode a signal peptide. Thesignal peptide can be an immunoglobulin (Ig) signal peptide, forexample, but not limited to, an IgG signal peptide and an IgE signalpeptide.

Arrangement of the Recombinant Nucleic Acid Sequence Construct

As described above, the recombinant nucleic acid sequence can includeone or more recombinant nucleic acid sequence constructs, in which eachrecombinant nucleic acid sequence construct can include one or morecomponents. The one or more components are described in detail above.The one or more components, when included in the recombinant nucleicacid sequence construct, can be arranged in any order relative to oneanother. In some embodiments, the one or more components can be arrangedin the recombinant nucleic acid sequence construct as described below.

(12) Arrangement 1

In one arrangement, a first recombinant nucleic acid sequence constructcan include the heterologous nucleic acid sequence encoding the heavychain polypeptide and a second recombinant nucleic acid sequenceconstruct can include the heterologous nucleic acid sequence encodingthe light chain polypeptide.

The first recombinant nucleic acid sequence construct can be placed in avector. The second recombinant nucleic acid sequence construct can beplaced in a second or separate vector. Placement of the recombinantnucleic acid sequence construct into the vector is described in moredetail below.

The first recombinant nucleic acid sequence construct can also includethe promoter, intron, transcription termination region, initiationcodon, termination codon, and/or polyadenylation signal. The firstrecombinant nucleic acid sequence construct can further include theleader sequence, in which the leader sequence is located upstream (or5′) of the heterologous nucleic acid sequence encoding the heavy chainpolypeptide. Accordingly, the signal peptide encoded by the leadersequence can be linked by a peptide bond to the heavy chain polypeptide.

The second recombinant nucleic acid sequence construct can also includethe promoter, initiation codon, termination codon, and polyadenylationsignal. The second recombinant nucleic acid sequence construct canfurther include the leader sequence, in which the leader sequence islocated upstream (or 5′) of the heterologous nucleic acid sequenceencoding the light chain polypeptide. Accordingly, the signal peptideencoded by the leader sequence can be linked by a peptide bond to thelight chain polypeptide.

Accordingly, one example of arrangement 1 can include the first vector(and thus first recombinant nucleic acid sequence construct) encodingthe heavy chain polypeptide that includes VH and CH1, and the secondvector (and thus second recombinant nucleic acid sequence construct)encoding the light chain polypeptide that includes VL and CL. A secondexample of arrangement 1 can include the first vector (and thus firstrecombinant nucleic acid sequence construct) encoding the heavy chainpolypeptide that includes VH, CH1, hinge region, CH2, and CH3, and thesecond vector (and thus second recombinant nucleic acid sequenceconstruct) encoding the light chain polypeptide that includes VL and CL.

(13) Arrangement 2

In a second arrangement, the recombinant nucleic acid sequence constructcan include the heterologous nucleic acid sequence encoding the heavychain polypeptide and the heterologous nucleic acid sequence encodingthe light chain polypeptide. The heterologous nucleic acid sequenceencoding the heavy chain polypeptide can be positioned upstream (or 5′)of the heterologous nucleic acid sequence encoding the light chainpolypeptide. Alternatively, the heterologous nucleic acid sequenceencoding the light chain polypeptide can be positioned upstream (or 5′)of the heterologous nucleic acid sequence encoding the heavy chainpolypeptide.

The recombinant nucleic acid sequence construct can be placed in thevector as described in more detail below.

The recombinant nucleic acid sequence construct can include theheterologous nucleic acid sequence encoding the protease cleavage siteand/or the linker sequence. If included in the recombinant nucleic acidsequence construct, the heterologous nucleic acid sequence encoding theprotease cleavage site can be positioned between the heterologousnucleic acid sequence encoding the heavy chain polypeptide and theheterologous nucleic acid sequence encoding the light chain polypeptide.Accordingly, the protease cleavage site allows for separation of theheavy chain polypeptide and the light chain polypeptide into distinctpolypeptides upon expression. In other embodiments, if the linkersequence is included in the recombinant nucleic acid sequence construct,then the linker sequence can be positioned between the heterologousnucleic acid sequence encoding the heavy chain polypeptide and theheterologous nucleic acid sequence encoding the light chain polypeptide.

The recombinant nucleic acid sequence construct can also include thepromoter, intron, transcription termination region, initiation codon,termination codon, and/or polyadenylation signal. The recombinantnucleic acid sequence construct can include one or more promoters. Therecombinant nucleic acid sequence construct can include two promoterssuch that one promoter can be associated with the heterologous nucleicacid sequence encoding the heavy chain polypeptide and the secondpromoter can be associated with the heterologous nucleic acid sequenceencoding the light chain polypeptide. In still other embodiments, therecombinant nucleic acid sequence construct can include one promoterthat is associated with the heterologous nucleic acid sequence encodingthe heavy chain polypeptide and the heterologous nucleic acid sequenceencoding the light chain polypeptide.

The recombinant nucleic acid sequence construct can further include twoleader sequences, in which a first leader sequence is located upstream(or 5′) of the heterologous nucleic acid sequence encoding the heavychain polypeptide and a second leader sequence is located upstream (or5′) of the heterologous nucleic acid sequence encoding the light chainpolypeptide. Accordingly, a first signal peptide encoded by the firstleader sequence can be linked by a peptide bond to the heavy chainpolypeptide and a second signal peptide encoded by the second leadersequence can be linked by a peptide bond to the light chain polypeptide.

Accordingly, one example of arrangement 2 can include the vector (andthus recombinant nucleic acid sequence construct) encoding the heavychain polypeptide that includes VH and CH1, and the light chainpolypeptide that includes VL and CL, in which the linker sequence ispositioned between the heterologous nucleic acid sequence encoding theheavy chain polypeptide and the heterologous nucleic acid sequenceencoding the light chain polypeptide.

A second example of arrangement of 2 can include the vector (and thusrecombinant nucleic acid sequence construct) encoding the heavy chainpolypeptide that includes VH and CH1, and the light chain polypeptidethat includes VL and CL, in which the heterologous nucleic acid sequenceencoding the protease cleavage site is positioned between theheterologous nucleic acid sequence encoding the heavy chain polypeptideand the heterologous nucleic acid sequence encoding the light chainpolypeptide.

A third example of arrangement 2 can include the vector (and thusrecombinant nucleic acid sequence construct) encoding the heavy chainpolypeptide that includes VH, CH1, hinge region, CH2, and CH3, and thelight chain polypeptide that includes VL and CL, in which the linkersequence is positioned between the heterologous nucleic acid sequenceencoding the heavy chain polypeptide and the heterologous nucleic acidsequence encoding the light chain polypeptide.

A forth example of arrangement of 2 can include the vector (and thusrecombinant nucleic acid sequence construct) encoding the heavy chainpolypeptide that includes VH, CH1, hinge region, CH2, and CH3, and thelight chain polypeptide that includes VL and CL, in which theheterologous nucleic acid sequence encoding the protease cleavage siteis positioned between the heterologous nucleic acid sequence encodingthe heavy chain polypeptide and the heterologous nucleic acid sequenceencoding the light chain polypeptide.

Expression from the Recombinant Nucleic Acid Sequence Construct

As described above, the recombinant nucleic acid sequence construct caninclude, amongst the one or more components, the heterologous nucleicacid sequence encoding the heavy chain polypeptide and/or theheterologous nucleic acid sequence encoding the light chain polypeptide.Accordingly, the recombinant nucleic acid sequence construct canfacilitate expression of the heavy chain polypeptide and/or the lightchain polypeptide.

When arrangement 1 as described above is utilized, the first recombinantnucleic acid sequence construct can facilitate the expression of theheavy chain polypeptide and the second recombinant nucleic acid sequenceconstruct can facilitate expression of the light chain polypeptide. Whenarrangement 2 as described above is utilized, the recombinant nucleicacid sequence construct can facilitate the expression of the heavy chainpolypeptide and the light chain polypeptide.

Upon expression, for example, but not limited to, in a cell, organism,or mammal, the heavy chain polypeptide and the light chain polypeptidecan assemble into the synthetic antibody. In particular, the heavy chainpolypeptide and the light chain polypeptide can interact with oneanother such that assembly results in the synthetic antibody beingcapable of binding the antigen. In other embodiments, the heavy chainpolypeptide and the light chain polypeptide can interact with oneanother such that assembly results in the synthetic antibody being moreimmunogenic as compared to an antibody not assembled as describedherein. In still other embodiments, the heavy chain polypeptide and thelight chain polypeptide can interact with one another such that assemblyresults in the synthetic antibody being capable of eliciting or inducingan immune response against the antigen.

Vector

The recombinant nucleic acid sequence construct described above can beplaced in one or more vectors. The one or more vectors can contain anorigin of replication. The one or more vectors can be a plasmid,bacteriophage, bacterial artificial chromosome or yeast artificialchromosome. The one or more vectors can be either a self-replicationextra chromosomal vector, or a vector which integrates into a hostgenome.

Vectors include, but are not limited to, plasmids, expression vectors,recombinant viruses, any form of recombinant “naked DNA” vector, and thelike. A “vector” comprises a nucleic acid which can infect, transfect,transiently or permanently transduce a cell. It will be recognized thata vector can be a naked nucleic acid, or a nucleic acid complexed withprotein or lipid. The vector optionally comprises viral or bacterialnucleic acids and/or proteins, and/or membranes (e.g., a cell membrane,a viral lipid envelope, etc.). Vectors include, but are not limited toreplicons (e.g., RNA replicons, bacteriophages) to which fragments ofDNA may be attached and become replicated. Vectors thus include, but arenot limited to RNA, autonomous self-replicating circular or linear DNAor RNA (e.g., plasmids, viruses, and the like, see, e.g., U.S. Pat. No.5,217,879), and include both the expression and non-expression plasmids.In some embodiments, the vector includes linear DNA, enzymatic DNA orsynthetic DNA. Where a recombinant microorganism or cell culture isdescribed as hosting an “expression vector” this includes bothextrachromosomal circular and linear DNA and DNA that has beenincorporated into the host chromosome(s). Where a vector is beingmaintained by a host cell, the vector may either be stably replicated bythe cells during mitosis as an autonomous structure, or is incorporatedwithin the host's genome.

The one or more vectors can be a heterologous expression construct,which is generally a plasmid that is used to introduce a specific geneinto a target cell. Once the expression vector is inside the cell, theheavy chain polypeptide and/or light chain polypeptide that are encodedby the recombinant nucleic acid sequence construct is produced by thecellular-transcription and translation machinery ribosomal complexes.The one or more vectors can express large amounts of stable messengerRNA, and therefore proteins.

(14) Expression Vector

The one or more vectors can be a circular plasmid or a linear nucleicacid. The circular plasmid and linear nucleic acid are capable ofdirecting expression of a particular nucleotide sequence in anappropriate subject cell. The one or more vectors comprising therecombinant nucleic acid sequence construct may be chimeric, meaningthat at least one of its components is heterologous with respect to atleast one of its other components.

(15) Plasmid

The one or more vectors can be a plasmid. The plasmid may be useful fortransfecting cells with the recombinant nucleic acid sequence construct.The plasmid may be useful for introducing the recombinant nucleic acidsequence construct into the subject. The plasmid may also comprise aregulatory sequence, which may be well suited for gene expression in acell into which the plasmid is administered.

The plasmid may also comprise a mammalian origin of replication in orderto maintain the plasmid extrachromosomally and produce multiple copiesof the plasmid in a cell. The plasmid may be pVAX1, pCEP4 or pREP4 fromInvitrogen (San Diego, Calif.), which may comprise the Epstein Barrvirus origin of replication and nuclear antigen EBNA-1 coding region,which may produce high copy episomal replication without integration.The backbone of the plasmid may be pAV0242. The plasmid may be areplication defective adenovirus type 5 (Ad5) plasmid.

The plasmid may be pSE420 (Invitrogen, San Diego, Calif.), which may beused for protein production in Escherichia coli (E. coli). The plasmidmay also be pYES2 (Invitrogen, San Diego, Calif.), which may be used forprotein production in Saccharomyces cerevisiae strains of yeast. Theplasmid may also be of the MAXBAC™ complete baculovirus expressionsystem (Invitrogen, San Diego, Calif.), which may be used for proteinproduction in insect cells. The plasmid may also be pcDNAI or pcDNA3(Invitrogen, San Diego, Calif.), which may be used for proteinproduction in mammalian cells such as Chinese hamster ovary (CHO) cells.

(16) RNA

In one embodiment, the nucleic acid is an RNA molecule. In oneembodiment, the RNA molecule is transcribed from a DNA sequencedescribed herein. For example, in some embodiments, the RNA molecule isencoded by a DNA sequence at least 90% homologous to one of SEQ ID NO:3. In another embodiment, the nucleotide sequence comprises an RNAsequence transcribed by a DNA sequence encoding a polypeptide sequenceat least 90% homologous to one of SEQ ID NO 2, or a variant thereof or afragment thereof. Accordingly, in one embodiment, the invention providesan RNA molecule encoding one or more of the MAbs or DMAbs. The RNA maybe plus-stranded. Accordingly, in some embodiments, the RNA molecule canbe translated by cells without needing any intervening replication stepssuch as reverse transcription. A RNA molecule useful with the inventionmay have a 5′ cap (e.g. a 7-methylguanosine). This cap can enhance invivo translation of the RNA. The 5′ nucleotide of a RNA molecule usefulwith the invention may have a 5′ triphosphate group. In a capped RNAthis may be linked to a 7-methylguanosine via a 5′-to-5′ bridge. A RNAmolecule may have a 3′ poly-A tail. It may also include a poly-Apolymerase recognition sequence (e.g. AAUAAA) near its 3′ end. A RNAmolecule useful with the invention may be single-stranded. A RNAmolecule useful with the invention may comprise synthetic RNA. In someembodiments, the RNA molecule is a naked RNA molecule. In oneembodiment, the RNA molecule is comprised within a vector.

In one embodiment, the RNA has 5′ and 3′ UTRs. In one embodiment, the 5′UTR is between zero and 3000 nucleotides in length. The length of 5′ and3′ UTR sequences to be added to the coding region can be altered bydifferent methods, including, but not limited to, designing primers forPCR that anneal to different regions of the UTRs. Using this approach,one of ordinary skill in the art can modify the 5′ and 3′ UTR lengthsrequired to achieve optimal translation efficiency followingtransfection of the transcribed RNA.

The 5′ and 3′ UTRs can be the naturally occurring, endogenous 5′ and 3′UTRs for the gene of interest. Alternatively, UTR sequences that are notendogenous to the gene of interest can be added by incorporating the UTRsequences into the forward and reverse primers or by any othermodifications of the template. The use of UTR sequences that are notendogenous to the gene of interest can be useful for modifying thestability and/or translation efficiency of the RNA. For example, it isknown that AU-rich elements in 3′ UTR sequences can decrease thestability of RNA. Therefore, 3′ UTRs can be selected or designed toincrease the stability of the transcribed RNA based on properties ofUTRs that are well known in the art.

In one embodiment, the 5′ UTR can contain the Kozak sequence of theendogenous gene. Alternatively, when a 5′ UTR that is not endogenous tothe gene of interest is being added by PCR as described above, aconsensus Kozak sequence can be redesigned by adding the 5′ UTRsequence. Kozak sequences can increase the efficiency of translation ofsome RNA transcripts, but does not appear to be required for all RNAs toenable efficient translation. The requirement for Kozak sequences formany RNAs is known in the art. In other embodiments, the 5′ UTR can bederived from an RNA virus whose RNA genome is stable in cells. In otherembodiments, various nucleotide analogues can be used in the 3′ or 5′UTR to impede exonuclease degradation of the RNA.

In one embodiment, the RNA has both a cap on the 5′ end and a 3′ poly(A)tail which determine ribosome binding, initiation of translation andstability of RNA in the cell.

In one embodiment, the RNA is a nucleoside-modified RNA.Nucleoside-modified RNA have particular advantages over non-modifiedRNA, including for example, increased stability, low or absent innateimmunogenicity, and enhanced translation.

(17) Circular and Linear Vector

The one or more vectors may be circular plasmid, which may transform atarget cell by integration into the cellular genome or existextrachromosomally (e.g., autonomous replicating plasmid with an originof replication). The vector can be pVAX, pcDNA3.0, or provax, or anyother expression vector capable of expressing the heavy chainpolypeptide and/or light chain polypeptide encoded by the recombinantnucleic acid sequence construct.

Also provided herein is a linear nucleic acid, or linear expressioncassette (“LEC”), that is capable of being efficiently delivered to asubject via electroporation and expressing the heavy chain polypeptideand/or light chain polypeptide encoded by the recombinant nucleic acidsequence construct. The LEC may be any linear DNA devoid of anyphosphate backbone. The LEC may not contain any antibiotic resistancegenes and/or a phosphate backbone. The LEC may not contain other nucleicacid sequences unrelated to the desired gene expression.

The LEC may be derived from any plasmid capable of being linearized. Theplasmid may be capable of expressing the heavy chain polypeptide and/orlight chain polypeptide encoded by the recombinant nucleic acid sequenceconstruct. The plasmid can be pNP (Puerto Rico/34) or pM2 (NewCaledonia/99). The plasmid may be WLV009, pVAX, pcDNA3.0, or provax, orany other expression vector capable of expressing the heavy chainpolypeptide and/or light chain polypeptide encoded by the recombinantnucleic acid sequence construct.

The LEC can be perM2. The LEC can be perNP. perNP and perMR can bederived from pNP (Puerto Rico/34) and pM2 (New Caledonia/99),respectively.

(18) Viral Vectors

In one embodiment, viral vectors are provided herein which are capableof delivering a nucleic acid of the invention to a cell. The expressionvector may be provided to a cell in the form of a viral vector. Viralvector technology is well known in the art and is described, forexample, in Sambrook et al. (2001), and in Ausubel et al. (1997), and inother virology and molecular biology manuals. Viruses, which are usefulas vectors include, but are not limited to, retroviruses, adenoviruses,adeno-associated viruses, herpes viruses, and lentiviruses. In general,a suitable vector contains an origin of replication functional in atleast one organism, a promoter sequence, convenient restrictionendonuclease sites, and one or more selectable markers. (See, e.g., WO01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193. Viral vectors, andespecially retroviral vectors, have become the most widely used methodfor inserting genes into mammalian, e.g., human cells. Other viralvectors can be derived from lentivirus, poxviruses, herpes simplex virusI, adenoviruses and adeno-associated viruses, and the like. See, forexample, U.S. Pat. Nos. 5,350,674 and 5,585,362.

(19) Method of Preparing the Vector

Provided herein is a method for preparing the one or more vectors inwhich the recombinant nucleic acid sequence construct has been placed.After the final subcloning step, the vector can be used to inoculate acell culture in a large-scale fermentation tank, using known methods inthe art.

In other embodiments, after the final subcloning step, the vector can beused with one or more electroporation (EP) devices. The EP devices aredescribed below in more detail.

The one or more vectors can be formulated or manufactured using acombination of known devices and techniques, but preferably they aremanufactured using a plasmid manufacturing technique that is describedin a licensed, co-pending U.S. provisional application U.S. Ser. No.60/939,792, which was filed on May 23, 2007. In some examples, the DNAplasmids described herein can be formulated at concentrations greaterthan or equal to 10 mg/mL. The manufacturing techniques also include orincorporate various devices and protocols that are commonly known tothose of ordinary skill in the art, in addition to those described inU.S. Ser. No. 60/939,792, including those described in a licensedpatent, U.S. Pat. No. 7,238,522, which issued on Jul. 3, 2007. Theabove-referenced application and patent, U.S. Ser. No. 60/939,792 andU.S. Pat. No. 7,238,522, respectively, are hereby incorporated in theirentirety.

4. Antibody

As described above, the recombinant nucleic acid sequence can encode theantibody, a fragment thereof, a variant thereof, or a combinationthereof. The antibody can bind or react with the antigen, which isdescribed in more detail below.

The antibody may comprise a heavy chain and a light chaincomplementarity determining region (“CDR”) set, respectively interposedbetween a heavy chain and a light chain framework (“FR”) set whichprovide support to the CDRs and define the spatial relationship of theCDRs relative to each other. The CDR set may contain three hypervariableregions of a heavy or light chain V region. Proceeding from theN-terminus of a heavy or light chain, these regions are denoted as“CDR1,” “CDR2,” and “CDR3,” respectively. An antigen-binding site,therefore, may include six CDRs, comprising the CDR set from each of aheavy and a light chain V region.

The proteolytic enzyme papain preferentially cleaves IgG molecules toyield several fragments, two of which (the F(ab) fragments) eachcomprise a covalent heterodimer that includes an intact antigen-bindingsite. The enzyme pepsin is able to cleave IgG molecules to provideseveral fragments, including the F(ab′)2 fragment, which comprises bothantigen-binding sites. Accordingly, the antibody can be the Fab orF(ab′)2. The Fab can include the heavy chain polypeptide and the lightchain polypeptide. The heavy chain polypeptide of the Fab can includethe VH region and the CH1 region. The light chain of the Fab can includethe VL region and CL region.

The antibody can be an immunoglobulin (Ig). The Ig can be, for example,IgA, IgM, IgD, IgE, and IgG. The immunoglobulin can include the heavychain polypeptide and the light chain polypeptide. The heavy chainpolypeptide of the immunoglobulin can include a VH region, a CH1 region,a hinge region, a CH2 region, and a CH3 region. The light chainpolypeptide of the immunoglobulin can include a VL region and CL region.

The antibody can be a polyclonal or monoclonal antibody. The antibodycan be a chimeric antibody, a single chain antibody, an affinity maturedantibody, a human antibody, a humanized antibody, or a fully humanantibody. The humanized antibody can be an antibody from a non-humanspecies that binds the desired antigen having one or morecomplementarity determining regions (CDRs) from the non-human speciesand framework regions from a human immunoglobulin molecule.

The antibody can be a bispecific antibody as described below in moredetail. The antibody can be a bifunctional antibody as also describedbelow in more detail.

As described above, the antibody can be generated in the subject uponadministration of the composition to the subject. The antibody may havea half-life within the subject. In some embodiments, the antibody may bemodified to extend or shorten its half-life within the subject. Suchmodifications are described below in more detail.

The antibody can be defucosylated as described in more detail below.

The antibody may be modified to reduce or prevent antibody-dependentenhancement (ADE) of disease associated with the antigen as described inmore detail below.

Bispecific Antibody

The recombinant nucleic acid sequence can encode a bispecific antibody,a fragment thereof, a variant thereof, or a combination thereof. Thebispecific antibody can bind or react with two antigens, for example,two of the antigens described below in more detail. The bispecificantibody can be comprised of fragments of two of the antibodiesdescribed herein, thereby allowing the bispecific antibody to bind orreact with two desired target molecules, which may include the antigen,which is described below in more detail, a ligand, including a ligandfor a receptor, a receptor, including a ligand-binding site on thereceptor, a ligand-receptor complex, and a marker.

The invention provides novel bispecific antibodies comprising a firstantigen-binding site that specifically binds to a first target and asecond antigen-binding site that specifically binds to a second target,with particularly advantageous properties such as producibility,stability, binding affinity, biological activity, specific targeting ofcertain T cells, targeting efficiency and reduced toxicity. In someinstances, there are bispecific antibodies, wherein the bispecificantibody binds to the first target with high affinity and to the secondtarget with low affinity. In other instances, there are bispecificantibodies, wherein the bispecific antibody binds to the first targetwith low affinity and to the second target with high affinity. In otherinstances, there are bispecific antibodies, wherein the bispecificantibody binds to the first target with a desired affinity and to thesecond target with a desired affinity.

In one embodiment, the bispecific antibody is a bivalent antibodycomprising a) a first light chain and a first heavy chain of an antibodyspecifically binding to a first antigen, and b) a second light chain anda second heavy chain of an antibody specifically binding to a secondantigen.

In some embodiments, one of the binding sites of an antibody moleculeaccording to the invention is able to bind a T-cell specific receptormolecule and/or a natural killer cell (NK cell) specific receptormolecule. A T-cell specific receptor is the so called “T-cell receptor”(TCRs), which allows a T cell to bind to and, if additional signals arepresent, to be activated by and respond to an epitope/antigen presentedby another cell called the antigen-presenting cell or APC. The T cellreceptor is known to resemble a Fab fragment of a naturally occurringimmunoglobulin. It is generally monovalent, encompassing α- andβ-chains, in some embodiments, it encompasses γ-chains and δ-chains(supra). Accordingly, in some embodiments, the TCR is TCR (alpha/beta)and in some embodiments, it is TCR (gamma/delta). The T cell receptorforms a complex with the CD3 T-Cell co-receptor. CD3 is a proteincomplex and is composed of four distinct chains. In mammals, the complexcontains a CD3γ chain, a CD36 chain, and two CD3E chains. These chainsassociate with a molecule known as the T cell receptor (TCR) and theζ-chain to generate an activation signal in T lymphocytes. Hence, insome embodiments, a T-cell specific receptor is the CD3 T-Cellco-receptor. In some embodiments, a T-cell specific receptor is CD28, aprotein that is also expressed on T cells. CD28 can provideco-stimulatory signals, which are required for T cell activation. CD28plays important roles in T-cell proliferation and survival, cytokineproduction, and T-helper type-2 development. Yet a further example of aT-cell specific receptor is CD134, also termed Ox40. CD134/OX40 is beingexpressed after 24 to 72 hours following activation and can be taken todefine a secondary costimulatory molecule. Another example of a T-cellreceptor is 4-1 BB capable of binding to 4-1 BB-Ligand on antigenpresenting cells (APCs), whereby a costimulatory signal for the T cellis generated. Another example of a receptor predominantly found onT-cells is CDS, which is also found on B cells at low levels. A furtherexample of a receptor modifying T cell functions is CD95, also known asthe Fas receptor, which mediates apoptotic signaling by Fas-ligandexpressed on the surface of other cells. CD95 has been reported tomodulate TCR/CD3-driven signaling pathways in resting T lymphocytes.

An example of a NK cell specific receptor molecule is CD16, a lowaffinity Fc receptor and NKG2D. An example of a receptor molecule thatis present on the surface of both T cells and natural killer (NK) cellsis CD2 and further members of the CD2-superfamily. CD2 is able to act asa co-stimulatory molecule on T and NK cells.

In some embodiments, the first binding site of the antibody moleculebinds a MAYV antigen and the second binding site binds a T cell specificreceptor molecule and/or a natural killer (NK) cell specific receptormolecule.

Bifunctional Antibody

The recombinant nucleic acid sequence can encode a bifunctionalantibody, a fragment thereof, a variant thereof, or a combinationthereof. The bifunctional antibody can bind or react with the antigendescribed below. The bifunctional antibody can also be modified toimpart an additional functionality to the antibody beyond recognition ofand binding to the antigen. Such a modification can include, but is notlimited to, coupling to factor H or a fragment thereof. Factor H is asoluble regulator of complement activation and thus, may contribute toan immune response via complement-mediated lysis (CML).

Extension of Antibody Half-Life

As described above, the antibody may be modified to extend or shortenthe half-life of the antibody in the subject. The modification mayextend or shorten the half-life of the antibody in the serum of thesubject.

The modification may be present in a constant region of the antibody.The modification may be one or more amino acid substitutions in aconstant region of the antibody that extend the half-life of theantibody as compared to a half-life of an antibody not containing theone or more amino acid substitutions. The modification may be one ormore amino acid substitutions in the CH2 domain of the antibody thatextend the half-life of the antibody as compared to a half-life of anantibody not containing the one or more amino acid substitutions.

In some embodiments, the one or more amino acid substitutions in theconstant region may include replacing a methionine residue in theconstant region with a tyrosine residue, a serine residue in theconstant region with a threonine residue, a threonine residue in theconstant region with a glutamate residue, or any combination thereof,thereby extending the half-life of the antibody.

In other embodiments, the one or more amino acid substitutions in theconstant region may include replacing a methionine residue in the CH2domain with a tyrosine residue, a serine residue in the CH2 domain witha threonine residue, a threonine residue in the CH2 domain with aglutamate residue, or any combination thereof, thereby extending thehalf-life of the antibody.

Defucosylation

The recombinant nucleic acid sequence can encode an antibody that is notfucosylated (i.e., a defucosylated antibody or a non-fucosylatedantibody), a fragment thereof, a variant thereof, or a combinationthereof. Fucosylation includes the addition of the sugar fucose to amolecule, for example, the attachment of fucose to N-glycans, O-glycansand glycolipids. Accordingly, in a defucosylated antibody, fucose is notattached to the carbohydrate chains of the constant region. In turn,this lack of fucosylation may improve FcγRIIIa binding and antibodydirected cellular cytotoxic (ADCC) activity by the antibody as comparedto the fucosylated antibody. Therefore, in some embodiments, thenon-fucosylated antibody may exhibit increased ADCC activity as comparedto the fucosylated antibody.

The antibody may be modified so as to prevent or inhibit fucosylation ofthe antibody. In some embodiments, such a modified antibody may exhibitincreased ADCC activity as compared to the unmodified antibody. Themodification may be in the heavy chain, light chain, or a combinationthereof. The modification may be one or more amino acid substitutions inthe heavy chain, one or more amino acid substitutions in the lightchain, or a combination thereof.

Reduced ADE Response

The antibody may be modified to reduce or prevent antibody-dependentenhancement (ADE) of disease associated with the antigen, but stillneutralize the antigen.

In some embodiments, the antibody may be modified to include one or moreamino acid substitutions that reduce or prevent binding of the antibodyto FcγR1a. The one or more amino acid substitutions may be in theconstant region of the antibody. The one or more amino acidsubstitutions may include replacing a leucine residue with an alanineresidue in the constant region of the antibody, i.e., also known hereinas LA, LA mutation or LA substitution. The one or more amino acidsubstitutions may include replacing two leucine residues, each with analanine residue, in the constant region of the antibody and also knownherein as LALA, LALA mutation, or LALA substitution. The presence of theLALA substitutions may prevent or block the antibody from binding toFcγR1a, and thus, the modified antibody does not enhance or cause ADE ofdisease associated with the antigen, but still neutralizes the antigen.

Monoclonal Antibodies

In one embodiment, the invention provides anti-MAYV antibodies. Theantibodies may be intact monoclonal antibodies, and immunologicallyactive fragments (e.g., a Fab or (Fab)₂ fragment), a monoclonal antibodyheavy chain, or a monoclonal antibody light chain.

The antibody may comprise a heavy chain and a light chaincomplementarity determining region (“CDR”) set, respectively interposedbetween a heavy chain and a light chain framework (“FR”) set whichprovide support to the CDRs and define the spatial relationship of theCDRs relative to each other. The CDR set may contain three hypervariableregions of a heavy or light chain V region. Proceeding from theN-terminus of a heavy or light chain, these regions are denoted as“CDR1,” “CDR2,” and “CDR3,” respectively. An antigen-binding site,therefore, may include six CDRs, comprising the CDR set from each of aheavy and a light chain V region.

The antibody can be an immunoglobulin (Ig). The Ig can be, for example,IgA, IgM, IgD, IgE, and IgG. The immunoglobulin can include the heavychain polypeptide and the light chain polypeptide. The heavy chainpolypeptide of the immunoglobulin can include a VH region, a CH1 region,a hinge region, a CH2 region, and a CH3 region. The light chainpolypeptide of the immunoglobulin can include a VL region and CL region.

5. Antigen

The synthetic antibody is directed to the antigen or fragment or variantthereof. The antigen can be a nucleic acid sequence, an amino acidsequence, a polysaccharide or a combination thereof. The nucleic acidsequence can be DNA, RNA, cDNA, a variant thereof, a fragment thereof,or a combination thereof. The amino acid sequence can be a protein, apeptide, a variant thereof, a fragment thereof, or a combinationthereof. The polysaccharide can be a nucleic acid encodedpolysaccharide.

The antigen can be from a virus. The antigen can be associated withviral infection. In one embodiment, the antigen can be associated withMiddle East Respiratory Syndrome coronavirus (MERS-CoV) infection.

In one embodiment, a synthetic antibody of the invention targets two ormore antigens. In one embodiment, at least one antigen of a bispecificantibody is selected from the antigens described herein. In oneembodiment, the two or more antigens are selected from the antigensdescribed herein.

6. Excipients and Other Components of the Composition

The composition may further comprise a pharmaceutically acceptableexcipient. The pharmaceutically acceptable excipient can be functionalmolecules such as vehicles, carriers, or diluents. The pharmaceuticallyacceptable excipient can be a transfection facilitating agent, which caninclude surface active agents, such as immune-stimulating complexes(ISCOMS), Freunds incomplete adjuvant, LPS analog includingmonophosphoryl lipid A, muramyl peptides, quinone analogs, vesicles suchas squalene and squalene, hyaluronic acid, lipids, liposomes, calciumions, viral proteins, polyanions, polycations, or nanoparticles, orother known transfection facilitating agents.

The transfection facilitating agent is a polyanion, polycation,including poly-L-glutamate (LGS), or lipid. The transfectionfacilitating agent is poly-L-glutamate, and the poly-L-glutamate may bepresent in the composition at a concentration less than 6 mg/ml. Thetransfection facilitating agent may also include surface active agentssuch as immune-stimulating complexes (ISCOMS), Freunds incompleteadjuvant, LPS analog including monophosphoryl lipid A, muramyl peptides,quinone analogs and vesicles such as squalene and squalene, andhyaluronic acid may also be used administered in conjunction with thecomposition. The composition may also include a transfectionfacilitating agent such as lipids, liposomes, including lecithinliposomes or other liposomes known in the art, as a DNA-liposome mixture(see for example WO9324640), calcium ions, viral proteins, polyanions,polycations, or nanoparticles, or other known transfection facilitatingagents. The transfection facilitating agent is a polyanion, polycation,including poly-L-glutamate (LGS), or lipid. Concentration of thetransfection agent in the vaccine is less than 4 mg/ml, less than 2mg/ml, less than 1 mg/ml, less than 0.750 mg/ml, less than 0.500 mg/ml,less than 0.250 mg/ml, less than 0.100 mg/ml, less than 0.050 mg/ml, orless than 0.010 mg/ml.

The composition may further comprise a genetic facilitator agent asdescribed in U.S. Ser. No. 021,579 filed Apr. 1, 1994, which is fullyincorporated by reference.

The composition may comprise DNA at quantities of from about 1 nanogramto 100 milligrams; about 1 microgram to about 10 milligrams; orpreferably about 0.1 microgram to about 10 milligrams; or morepreferably about 1 milligram to about 2 milligram. In some preferredembodiments, composition according to the present invention comprisesabout 5 nanogram to about 1000 micrograms of DNA. In some preferredembodiments, composition can contain about 10 nanograms to about 800micrograms of DNA. In some preferred embodiments, the composition cancontain about 0.1 to about 500 micrograms of DNA. In some preferredembodiments, the composition can contain about 1 to about 350 microgramsof DNA. In some preferred embodiments, the composition can contain about25 to about 250 micrograms, from about 100 to about 200 microgram, fromabout 1 nanogram to 100 milligrams; from about 1 microgram to about 10milligrams; from about 0.1 microgram to about 10 milligrams; from about1 milligram to about 2 milligram, from about 5 nanogram to about 1000micrograms, from about 10 nanograms to about 800 micrograms, from about0.1 to about 500 micrograms, from about 1 to about 350 micrograms, fromabout 25 to about 250 micrograms, from about 100 to about 200 microgramof DNA.

The composition can be formulated according to the mode ofadministration to be used. An injectable pharmaceutical composition canbe sterile, pyrogen free and particulate free. An isotonic formulationor solution can be used. Additives for isotonicity can include sodiumchloride, dextrose, mannitol, sorbitol, and lactose. The composition cancomprise a vasoconstriction agent. The isotonic solutions can includephosphate buffered saline. The composition can further comprisestabilizers including gelatin and albumin. The stabilizers can allow theformulation to be stable at room or ambient temperature for extendedperiods of time, including LGS or polycations or polyanions.

7. Method of Generating the Synthetic Antibody

The present invention also relates a method of generating the syntheticantibody. The method can include administering the composition to thesubject in need thereof by using the method of delivery described inmore detail below. Accordingly, the synthetic antibody is generated inthe subject or in vivo upon administration of the composition to thesubject.

The method can also include introducing the composition into one or morecells, and therefore, the synthetic antibody can be generated orproduced in the one or more cells. The method can further includeintroducing the composition into one or more tissues, for example, butnot limited to, skin and muscle, and therefore, the synthetic antibodycan be generated or produced in the one or more tissues.

8. Method of Identifying or Screening for the Antibody

The present invention further relates to a method of identifying orscreening for the antibody described above, which is reactive to orbinds the antigen described above. The method of identifying orscreening for the antibody can use the antigen in methodologies known inthose skilled in art to identify or screen for the antibody. Suchmethodologies can include, but are not limited to, selection of theantibody from a library (e.g., phage display) and immunization of ananimal followed by isolation and/or purification of the antibody.

9. Method of Delivery of the Composition

The present invention also relates to a method of delivering thecomposition to the subject in need thereof. The method of delivery caninclude, administering the composition to the subject. Administrationcan include, but is not limited to, DNA injection with and without invivo electroporation, liposome mediated delivery, and nanoparticlefacilitated delivery.

The mammal receiving delivery of the composition may be human, primate,non-human primate, cow, cattle, sheep, goat, antelope, bison, waterbuffalo, bison, bovids, deer, hedgehogs, elephants, llama, alpaca, mice,rats, and chicken.

The composition may be administered by different routes includingorally, parenterally, sublingually, transdermally, rectally,transmucosally, topically, via inhalation, via buccal administration,intrapleurally, intravenous, intraarterial, intraperitoneal,subcutaneous, intramuscular, intranasal, intranasal, intrathecal, andintraarticular or combinations thereof. For veterinary use, thecomposition may be administered as a suitably acceptable formulation inaccordance with normal veterinary practice. The veterinarian can readilydetermine the dosing regimen and route of administration that is mostappropriate for a particular animal. The composition may be administeredby traditional syringes, needleless injection devices, “microprojectilebombardment gone guns”, or other physical methods such aselectroporation (“EP”), “hydrodynamic method”, or ultrasound.

Electroporation

Administration of the composition via electroporation may beaccomplished using electroporation devices that can be configured todeliver to a desired tissue of a mammal, a pulse of energy effective tocause reversible pores to form in cell membranes, and preferable thepulse of energy is a constant current similar to a preset current inputby a user. The electroporation device may comprise an electroporationcomponent and an electrode assembly or handle assembly. Theelectroporation component may include and incorporate one or more of thevarious elements of the electroporation devices, including: controller,current waveform generator, impedance tester, waveform logger, inputelement, status reporting element, communication port, memory component,power source, and power switch. The electroporation may be accomplishedusing an in vivo electroporation device, for example CELLECTRA EP system(Inovio Pharmaceuticals, Plymouth Meeting, Pa.) or Elgen electroporator(Inovio Pharmaceuticals, Plymouth Meeting, Pa.) to facilitatetransfection of cells by the plasmid.

The electroporation component may function as one element of theelectroporation devices, and the other elements are separate elements(or components) in communication with the electroporation component. Theelectroporation component may function as more than one element of theelectroporation devices, which may be in communication with still otherelements of the electroporation devices separate from theelectroporation component. The elements of the electroporation devicesexisting as parts of one electromechanical or mechanical device may notlimited as the elements can function as one device or as separateelements in communication with one another. The electroporationcomponent may be capable of delivering the pulse of energy that producesthe constant current in the desired tissue, and includes a feedbackmechanism. The electrode assembly may include an electrode array havinga plurality of electrodes in a spatial arrangement, wherein theelectrode assembly receives the pulse of energy from the electroporationcomponent and delivers same to the desired tissue through theelectrodes. At least one of the plurality of electrodes is neutralduring delivery of the pulse of energy and measures impedance in thedesired tissue and communicates the impedance to the electroporationcomponent. The feedback mechanism may receive the measured impedance andcan adjust the pulse of energy delivered by the electroporationcomponent to maintain the constant current.

A plurality of electrodes may deliver the pulse of energy in adecentralized pattern. The plurality of electrodes may deliver the pulseof energy in the decentralized pattern through the control of theelectrodes under a programmed sequence, and the programmed sequence isinput by a user to the electroporation component. The programmedsequence may comprise a plurality of pulses delivered in sequence,wherein each pulse of the plurality of pulses is delivered by at leasttwo active electrodes with one neutral electrode that measuresimpedance, and wherein a subsequent pulse of the plurality of pulses isdelivered by a different one of at least two active electrodes with oneneutral electrode that measures impedance.

The feedback mechanism may be performed by either hardware or software.The feedback mechanism may be performed by an analog closed-loopcircuit. The feedback occurs every 50 μs, 20 μs, 10 μs or 1 μs, but ispreferably a real-time feedback or instantaneous (i.e., substantiallyinstantaneous as determined by available techniques for determiningresponse time). The neutral electrode may measure the impedance in thedesired tissue and communicates the impedance to the feedback mechanism,and the feedback mechanism responds to the impedance and adjusts thepulse of energy to maintain the constant current at a value similar tothe preset current. The feedback mechanism may maintain the constantcurrent continuously and instantaneously during the delivery of thepulse of energy.

Examples of electroporation devices and electroporation methods that mayfacilitate delivery of the composition of the present invention, includethose described in U.S. Pat. No. 7,245,963 by Draghia-Akli, et al., U.S.Patent Pub. 2005/0052630 submitted by Smith, et al., the contents ofwhich are hereby incorporated by reference in their entirety. Otherelectroporation devices and electroporation methods that may be used forfacilitating delivery of the composition include those provided inco-pending and co-owned U.S. patent application Ser. No. 11/874,072,filed Oct. 17, 2007, which claims the benefit under 35 USC 119(e) toU.S. Provisional Application Ser. No. 60/852,149, filed Oct. 17, 2006,and 60/978,982, filed Oct. 10, 2007, all of which are herebyincorporated in their entirety.

U.S. Pat. No. 7,245,963 by Draghia-Akli, et al. describes modularelectrode systems and their use for facilitating the introduction of abiomolecule into cells of a selected tissue in a body or plant. Themodular electrode systems may comprise a plurality of needle electrodes;a hypodermic needle; an electrical connector that provides a conductivelink from a programmable constant-current pulse controller to theplurality of needle electrodes; and a power source. An operator cangrasp the plurality of needle electrodes that are mounted on a supportstructure and firmly insert them into the selected tissue in a body orplant. The biomolecules are then delivered via the hypodermic needleinto the selected tissue. The programmable constant-current pulsecontroller is activated and constant-current electrical pulse is appliedto the plurality of needle electrodes. The applied constant-currentelectrical pulse facilitates the introduction of the biomolecule intothe cell between the plurality of electrodes. The entire content of U.S.Pat. No. 7,245,963 is hereby incorporated by reference.

U.S. Patent Pub. 2005/0052630 submitted by Smith, et al. describes anelectroporation device which may be used to effectively facilitate theintroduction of a biomolecule into cells of a selected tissue in a bodyor plant. The electroporation device comprises an electro-kinetic device(“EKD device”) whose operation is specified by software or firmware. TheEKD device produces a series of programmable constant-current pulsepatterns between electrodes in an array based on user control and inputof the pulse parameters, and allows the storage and acquisition ofcurrent waveform data. The electroporation device also comprises areplaceable electrode disk having an array of needle electrodes, acentral injection channel for an injection needle, and a removable guidedisk. The entire content of U.S. Patent Pub. 2005/0052630 is herebyincorporated by reference.

The electrode arrays and methods described in U.S. Pat. No. 7,245,963and U.S. Patent Pub. 2005/0052630 may be adapted for deep penetrationinto not only tissues such as muscle, but also other tissues or organs.Because of the configuration of the electrode array, the injectionneedle (to deliver the biomolecule of choice) is also insertedcompletely into the target organ, and the injection is administeredperpendicular to the target issue, in the area that is pre-delineated bythe electrodes The electrodes described in U.S. Pat. No. 7,245,963 andU.S. Patent Pub. 2005/005263 are preferably 20 mm long and 21 gauge.

Additionally, contemplated in some embodiments, that incorporateelectroporation devices and uses thereof, there are electroporationdevices that are those described in the following patents: U.S. Pat. No.5,273,525 issued Dec. 28, 1993, U.S. Pat. No. 6,110,161 issued Aug. 29,2000, U.S. Pat. No. 6,261,281 issued Jul. 17, 2001, and U.S. Pat. No.6,958,060 issued Oct. 25, 2005, and U.S. Pat. No. 6,939,862 issued Sep.6, 2005. Furthermore, patents covering subject matter provided in U.S.Pat. No. 6,697,669 issued Feb. 24, 2004, which concerns delivery of DNAusing any of a variety of devices, and U.S. Pat. No. 7,328,064 issuedFeb. 5, 2008, drawn to method of injecting DNA are contemplated herein.The above-patents are incorporated by reference in their entirety.

10. Method of Treatment

Also provided herein is a method of treating, protecting against, and/orpreventing disease in a subject in need thereof by generating thesynthetic antibody in the subject. The method can include administeringthe composition to the subject. Administration of the composition to thesubject can be done using the method of delivery described above.

In certain embodiments, the invention provides a method of treatingprotecting against, and/or preventing a Middle East Respiratory Syndromecoronavirus (MERS-CoV) infection. In one embodiment, the method treats,protects against, and/or prevents a disease associated with MERS-CoV.

Upon generation of the synthetic antibody in the subject, the syntheticantibody can bind to or react with the antigen. Such binding canneutralize the antigen, block recognition of the antigen by anothermolecule, for example, a protein or nucleic acid, and elicit or inducean immune response to the antigen, thereby treating, protecting against,and/or preventing the disease associated with the antigen in thesubject.

The synthetic antibody can treat, prevent, and/or protect againstdisease in the subject administered the composition. The syntheticantibody by binding the antigen can treat, prevent, and/or protectagainst disease in the subject administered the composition. Thesynthetic antibody can promote survival of the disease in the subjectadministered the composition. In one embodiment, the synthetic antibodycan provide increased survival of the disease in the subject over theexpected survival of a subject having the disease who has not beenadministered the synthetic antibody. In various embodiments, thesynthetic antibody can provide at least about a 1%, 2%, 3%, 4%, 5%, 6%,7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or a 100% increase in survival of thedisease in subjects administered the composition over the expectedsurvival in the absence of the composition. In one embodiment, thesynthetic antibody can provide increased protection against the diseasein the subject over the expected protection of a subject who has notbeen administered the synthetic antibody. In various embodiments, thesynthetic antibody can protect against disease in at least about 1%, 2%,3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of subjectsadministered the composition over the expected protection in the absenceof the composition.

The composition dose can be between 1 μg to 10 mg active component/kgbody weight/time, and can be 20 μg to 10 mg component/kg bodyweight/time. The composition can be administered every 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, or 31 days. The number of composition doses foreffective treatment can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

11. Use in Combination with Antibiotics

The present invention also provides a method of treating, protectingagainst, and/or preventing disease in a subject in need thereof byadministering a combination of the synthetic antibody and a therapeuticantibiotic agent.

The synthetic antibody and an antibiotic agent may be administered usingany suitable method such that a combination of the synthetic antibodyand antibiotic agent are both present in the subject. In one embodiment,the method may comprise administration of a first composition comprisinga synthetic antibody of the invention by any of the methods described indetail above and administration of a second composition comprising anantibiotic agent less than 1, less than 2, less than 3, less than 4,less than 5, less than 6, less than 7, less than 8, less than 9 or lessthan 10 days following administration of the synthetic antibody. In oneembodiment, the method may comprise administration of a firstcomposition comprising a synthetic antibody of the invention by any ofthe methods described in detail above and administration of a secondcomposition comprising an antibiotic agent more than 1, more than 2,more than 3, more than 4, more than 5, more than 6, more than 7, morethan 8, more than 9 or more than 10 days following administration of thesynthetic antibody. In one embodiment, the method may compriseadministration of a first composition comprising an antibiotic agent andadministration of a second composition comprising a synthetic antibodyof the invention by any of the methods described in detail above lessthan 1, less than 2, less than 3, less than 4, less than 5, less than 6,less than 7, less than 8, less than 9 or less than 10 days followingadministration of the antibiotic agent. In one embodiment, the methodmay comprise administration of a first composition comprising anantibiotic agent and administration of a second composition comprising asynthetic antibody of the invention by any of the methods described indetail above more than 1, more than 2, more than 3, more than 4, morethan 5, more than 6, more than 7, more than 8, more than 9 or more than10 days following administration of the antibiotic agent. In oneembodiment, the method may comprise administration of a firstcomposition comprising a synthetic antibody of the invention by any ofthe methods described in detail above and a second compositioncomprising an antibiotic agent concurrently. In one embodiment, themethod may comprise administration of a first composition comprising asynthetic antibody of the invention by any of the methods described indetail above and a second composition comprising an antibiotic agentconcurrently. In one embodiment, the method may comprise administrationof a single composition comprising a synthetic antibody of the inventionand an antibiotic agent.

Non-limiting examples of antibiotics that can be used in combinationwith the synthetic antibody of the invention include aminoglycosides(e.g., gentamicin, amikacin, tobramycin), quinolones (e.g.,ciprofloxacin, levofloxacin), cephalosporins (e.g., ceftazidime,cefepime, cefoperazone, cefpirome, ceftobiprole), antipseudomonalpenicillins: carboxypenicillins (e.g., carbenicillin and ticarcillin)and ureidopenicillins (e.g., mezlocillin, azlocillin, and piperacillin),carbapenems (e.g., meropenem, imipenem, doripenem), polymyxins (e.g.,polymyxin B and colistin) and monobactams (e.g., aztreonam).

The present invention has multiple aspects, illustrated by the followingnon-limiting examples.

12. Generation of Synthetic Antibodies In Vitro and Ex Vivo

In one embodiment, the synthetic antibody is generated in vitro or exvivo. For example, in one embodiment, a nucleic acid encoding asynthetic antibody can be introduced and expressed in an in vitro or exvivo cell. Methods of introducing and expressing genes into a cell areknown in the art. In the context of an expression vector, the vector canbe readily introduced into a host cell, e.g., mammalian, bacterial,yeast, or insect cell by any method in the art. For example, theexpression vector can be transferred into a host cell by physical,chemical, or biological means.

Physical methods for introducing a polynucleotide into a host cellinclude calcium phosphate precipitation, lipofection, particlebombardment, microinjection, electroporation, and the like. Methods forproducing cells comprising vectors and/or exogenous nucleic acids arewell-known in the art. See, for example, Sambrook et al. (2012,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,New York). A preferred method for the introduction of a polynucleotideinto a host cell is calcium phosphate transfection.

Biological methods for introducing a polynucleotide of interest into ahost cell include the use of DNA and RNA vectors. Viral vectors, andespecially retroviral vectors, have become the most widely used methodfor inserting genes into mammalian, e.g., human cells. Other viralvectors can be derived from lentivirus, poxviruses, herpes simplex virusI, adenoviruses and adeno-associated viruses, and the like. See, forexample, U.S. Pat. Nos. 5,350,674 and 5,585,362.

Chemical means for introducing a polynucleotide into a host cell includecolloidal dispersion systems, such as macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Anexemplary colloidal system for use as a delivery vehicle in vitro and invivo is a liposome (e.g., an artificial membrane vesicle).

In the case where a non-viral delivery system is utilized, an exemplarydelivery vehicle is a liposome. The use of lipid formulations iscontemplated for the introduction of the nucleic acids into a host cell(in vitro, ex vivo or in vivo). In another aspect, the nucleic acid maybe associated with a lipid. The nucleic acid associated with a lipid maybe encapsulated in the aqueous interior of a liposome, interspersedwithin the lipid bilayer of a liposome, attached to a liposome via alinking molecule that is associated with both the liposome and theoligonucleotide, entrapped in a liposome, complexed with a liposome,dispersed in a solution containing a lipid, mixed with a lipid, combinedwith a lipid, contained as a suspension in a lipid, contained orcomplexed with a micelle, or otherwise associated with a lipid. Lipid,lipid/DNA or lipid/expression vector associated compositions are notlimited to any particular structure in solution. For example, they maybe present in a bilayer structure, as micelles, or with a “collapsed”structure. They may also simply be interspersed in a solution, possiblyforming aggregates that are not uniform in size or shape. Lipids arefatty substances which may be naturally occurring or synthetic lipids.For example, lipids include the fatty droplets that naturally occur inthe cytoplasm as well as the class of compounds which contain long-chainaliphatic hydrocarbons and their derivatives, such as fatty acids,alcohols, amines, amino alcohols, and aldehydes.

13. Examples

The present invention is further illustrated in the following Examples.It should be understood that these Examples, while indicating preferredembodiments, of the invention, are given by way of illustration only.From the above discussion and these Examples, one skilled in the art canascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions. Thus, various modifications of the invention in addition tothose shown and described herein will be apparent to those skilled inthe art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims.

Example 1

Anti-Middle East Respiratory Syndrome coronavirus (MERS-CoV)(anti-MERS-CoV) “DNA monoclonal antibodies” (DMAb) can be generated viaintramuscular electroporation of plasmid DNA.

As described herein, an optimized, synthetic DNA vector platform (DMAb)to deliver encoded mAb heavy and light chains directly into skeletalmuscle was designed, employing the cells as biological factories thatwill secrete a functional antibody at detectable levels in systemiccirculation. DMAbs encoding anti-MERS-CoV mAbs that target the MERS-CoVviral proteins are developed (FIG. 3).

Monoclonal antibody (mAb) therapies have successfully been employed totreat a myriad of diseases. However, the labor and cost involved in thedevelopment and manufacturing of conventional protein mAbs haveprohibited their global use, especially against infectious diseases inlow resource countries. In response to this dilemma, gene transfermethods are being developed with the goal of delivering mAb encodingtransgenes in vivo, endowing the animal's own cells to function asantibody producing factories. One such technology is DNA-basedmonoclonal antibodies (dMAb™). Herein, plasmid DNA encoding for humanIgG is injected into the muscle. Electroporation immediately afterinjection enables pDNA to enter the target cells (myocytes). The dMAb™is expressed in the target cells and secreted. The goal is to achievesystemic mAb levels which confer protection against target pathogens.

Herein is described the advancement of dMAb™ technology fromproof-of-concept studies in small animals through to large animals,supporting the translation of the platform into the clinic forfirst-in-human studies. The precise optimization of the protocolemployed for the delivery of the pDNA to the muscle permits high serumlevels of dMAb™ to be achieved. The optimized delivery protocol combinesin vivo electroporation (EP; previously shown to enhance gene expressionup to 1000 fold), with an enhanced drug formulation. The formulationincludes an enzyme which is utilized in the clinic to enhance drugdispersion. This protocol was employed to dramatically increase thelocal (in the target tissue) and systemic (serum) levels of dMAb™ (FIG.1). The optimized delivery protocol translated to larger animals toachieve robust systemic levels of dMAbs™ in rhesus macaques (FIG. 2).

An enhanced pDNA delivery platform has been achieved by employing thecombination of EP and an optimized formulation to increase dMAb™expression.

This delivery platform has been employed to achieve robust serum-levelsof functional human antibody in mice, rabbits and non-human primates(NHPs). Data depicted herein support the translation of dMAb™ technologyinto humans.

pDNA is non-replicating, easy to rapidly manufacture, generates noanti-vector immunity, stable at ambient temperatures, has an excellentsafety profile and can be efficiently delivered.

dMAb™ technology offers an affordable and accessible platform to achieverapid mAb interventions.

Example 2

SEQ ID NO:1: nucleotide sequence of pGX9207, nucleic acid sequence of anexpression plasmid for expression of an Anti-MERS-CoV DMAb.

SEQ ID NO:2: amino acid sequence of an anti-MERS dMAB

SEQ ID NO:3: nucleotide sequence of an anti-MERS dMAB

SEQ ID NO:4: nucleotide sequence of an anti-MERS dMAB heavy chain

SEQ ID NO:5: amino acid sequence of an anti-MERS dMAB heavy chain

SEQ ID NO:6: nucleotide sequence of an anti-MERS dMAB light chain

SEQ ID NO:7: amino acid nucleotide sequence of an anti-MERS dMAB lightchain

It is understood that the foregoing detailed description andaccompanying examples are merely illustrative and are not to be taken aslimitations upon the scope of the invention, which is defined solely bythe appended claims and their equivalents.

Various changes and modifications to the disclosed embodiments, will beapparent to those skilled in the art. Such changes and modifications,including without limitation those relating to the chemical structures,substituents, derivatives, intermediates, syntheses, compositions,formulations, or methods of use of the invention, may be made withoutdeparting from the spirit and scope thereof.

What is claimed is:
 1. A nucleic acid molecule encoding one or moresynthetic antibodies, wherein the nucleic acid molecule comprises atleast one selected from the group consisting of: a) a nucleotidesequence encoding an anti-Middle East Respiratory Syncytial Coronavirus(MERS-CoV) synthetic antibody comprising the amino acid sequence of SEQID NO:2; and b) a nucleotide sequence encoding a fragment of ananti-MERS-CoV synthetic antibody comprising at least one selected froman anti-MERS dMAB heavy chain comprising the amino acid sequence of SEQID NO:5 and an anti-MERS dMAB light chain comprising the amino acidsequence of SEQ ID NO:7.
 2. The nucleic acid molecule of claim 1,wherein the one or more synthetic antibodies binds to a MERS-CoVantigen.
 3. The nucleic acid molecule of claim 1, further comprising anucleotide sequence encoding a cleavage domain.
 4. The nucleic acidmolecule of claim 1, comprising a nucleotide sequence at least 90%identical to at least one nucleotide sequence selected from the groupconsisting of SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:6.
 5. The nucleicacid molecule of claim 1, wherein the nucleotide sequence encodes aleader sequence.
 6. The nucleic acid molecule of claim 1, wherein thenucleic acid molecule comprises an expression vector.
 7. A compositioncomprising the nucleic acid molecule of claim
 6. 8. A compositioncomprising the nucleic acid molecule of claim 6, further comprising apharmaceutically acceptable excipient.